Insulin Allostery. The insulin hexamer is an allosteric protein that exhibits both positive and negative cooperativity and half-of-the-sites reactivity in ligand binding. This allosteric behaviour consists of two interrelated allosteric transitions designated LA0 and LB0, three interconverting allosteric conformation states (eq. 1),
                                          T            6                    ⁢                      ⟷                          L              0              A                                ⁢                      T            3                          ⁢                              R            3                    ⁢                      ⟷                          L              0              B                                ⁢                      R            6                                              (        1        )            designated T6, T3R3, and R6 and two classes of allosteric ligand binding sites designated as the phenolic pockets and the HisB10 anion sites. These allosteric sites are associated only with insulin subunits in the R conformation.
Insulin Hexamer Structures and Ligand Binding. The T- to R-transition of the insulin hexamer involves transformation of the first nine residues of the B chain from an extended conformation in the T-state to an α-helical conformation in the R-state. This coil-to-helix transition causes the N-terminal residue, PheB1, to undergo an ˜30 Å change in position. This conformational change creates hydrophobic pockets (the phenolic pockets) at the sub-unit interfaces (three in T3R3, and six in R6), and the new B-chain helices form 3-helix bundles (one in T3R3 and two in R6) with the bundle axis aligned along the hexamer three-fold symmetry axis. The HisB10 Zn2+ in each R3 unit is forced to change coordination geometry from octahedral to either tetrahedral (monodentate ligands) or pentahedran (bidentate ligands). Formation of the helix bundle creates a narrow hydrophobic tunnel in each R3 unit that extends from the surface ˜12 Å down to the HisB10 metal ion. This tunnel and the HisB10 Zn2+ ion form the anion binding site.
Hexamer Ligand Binding and Stability of Insulin Formulations. The in vivo role of the T to R transition is unknown. However, the addition of allosteric ligands (e.g. phenol and chloride ion) to insulin preparations is widely used. Hexamerization is driven by coordination of Zn2+ at the HisB10 sites to give T6, and the subsequent ligand-mediated transition of T6 to T3R3 and to R6 is known to greatly enhance the physical and chemical stability of the resulting formulations.
Ligand Binding and Long Acting Insulin Formulations. Although the conversion of T6 to T3R3 and R6 improves the stability of the preparation, the rate of absorption following subcutaneous injection of a soluble hexameric preparation is not much affected by the addition of phenol and chloride.
Putative events following injection of a soluble hexameric preparation. The small molecule ligands initially diffuse away from the protein. The affinity of the ligands for insulin may help to slow this process. On the other hand, the affinity of Zn2+ for e.g. albumin and the large effective space available for diffusion of the lipophilic phenol will tend to speed up the separation. In about 10-15 minutes after injection, the distribution of insulin species in the subcutaneous tissue will roughly correspond to that of a zinc-free insulin preparation at the same dilution. Then, the equilibrium distribution of species at this point will determine the observed absorption rate. In this regimen, absorption rates vary between about 1 hour (for rapid-acting insulin analogues, such as AspB28 human insulin) and about 4 hours (Co3+-hexamer).
Current Approaches Toward Slow Acting Insulins. The inherent limitation of the absorption half-life to about 4 hours for a soluble human insulin hexamer necessitates further modifications to obtain the desired protraction. Traditionally, this has been achieved by the use of preparations wherein the constituent insulin is in the form of a crystalline and/or amorphous precipitate. In this type of formulation, the dissolution of the precipitate in the subcutaneous depot becomes rate-limiting for the absorption. NPH and Ultralente belong to this category of insulin preparations where crystallization/precipitation is effected by the addition of protamine and excessive zinc ion, respectively.
Another approach involves the use of insulin derivatives where the net charge is increased to shift the isoelectric point, and hence the pH of minimum solubility, from about 5.5 to the physiological range. Such preparations may be injected as clear solutions at slightly acidic pH. The subsequent adjustment of the pH to neutral induces crystallization/precipitation in the subcutaneous depot and dissolution again becomes rate-limiting for the absorption. GlyA21ArgB31ArgB32 human insulin belongs to this category of insulin analogues.
Most recently, a series of soluble insulin derivatives with a hydrophobic moiety covalently attached to the side chain of LysB29 have been synthesized. These derivatives may show prolonged action profile due to various mechanisms including albumin binding (e.g. B29-Nε-myristoyl-des(B30) human insulin), extensive protein self-association and/or stickiness (e.g. B29-Nε-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin) induced by the attached hydrophobic group.